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Notes: The authors of this study describe mutations in a gene, Enigma (Egm), that increase lifespan and protect against oxidative stress. They mapped the gene and showed that it encodes a mitochontrial protein that is homologous to enzymes involved in the β-oxidation of fatty acids. To determine what other genes might be affected by loss of function of Egm, the authors performed an RNAi experiment using Drosophila Kc-167 cells. Double-stranded RNA was produced using the RiboMax™ Large Scale RNA Production System, and a portion of the Egm cDNA was amplified from that RNA. Some of the genes affected by the Egm RNA include a homolog of a mammalian enzyme known to be involved in fatty acid β-oxidation and enzymes involved in detoxification including glutathione-S-transferase and a cytochrome P450. (3668)

Notes: microRNAs (miRNAs) are a class of endogenous short RNAs that repress gene expression. To assess miRNA-directed translational silencing, in vitro reactions were performed. Translational gene silencing by miRNAs in vitro requires target mRNAs to possess a 7-methyl G cap and a poly(A) tail. In vitro transcription was performed with 5μg of linearized plasmids containing zero, two, four, or six miCXCR4 binding sites, one siCXCR4 binding site or Renilla luciferase (pRL-TK; control mRNA) using the RiboMax™ T7 Large-Scale RNA Production kit. The mRNAs were modified by 7-methyl-G capping and polyadenylation. Translation was performed using nuclease-treated rabbit reticulocyte lysate containing 0.025pmole of mRNA with miCXCR4 or siCXCR4 binding sites, 0.025pmole Renilla control RNA, and different ratios of CXCR4 siRNA. Reaction products were separated by SDS-PAGE and visualized and quantitated by PhosphorImager analysis. (3412)

Notes: The authors of this paper demonstrate that the pathways controlling lifespan and senescence are linked to the pathways controlling fertility in the honey bee. Using RNAi experiments, the authors demonstrate that vitellogenin protects honey bee workers from oxidative stress. Double-stranded RNA was synthesized from the vitellogenin clone AP4a5 using the RiboMax® T7 System. In additional experiments the authors assessed apoptosis in brain tissue sections using the DeadEND™ Fluorometric TUNEL system. (3636)

Notes: In this report, satellite RNA from the full-length RPV serotype (satRPV RNA) of Cereal yellow dwarf virus was transcribed in vitro using the RiboMAX™ Large Scale RNA Production System. Self-cleavage of the synthesized RNA was then induced by incubation with a cleavage buffer. Serial deletions were created in the wild-type satRPV cloned in pGEM®-3Zf(–) Vector using the Erase-a-Base® System. The mutants generated were tested to see how well they replicated with internal sections of satRPV sequence removed. (3080)

Notes: Several different sequences from a S. pneumoniae pA promoter region were cloned into the pSP-luc+ vector and screened for expression levels in in vitro transcription/translation systems. The RiboMAX™ SP6 Large Scale RNA Production System was used to transcribe luciferase encoding mRNAs. Luciferase mRNAs and plasmids were used as templates in high throughput inhibition studies in an S. pneumoniae S30 extract described in the paper as well as in Promega’s E. Coli S30 Extract System for Circular DNA and in Rabbit Reticulocyte Lysate. Promega amino acid mixtures were also used in these studies. (3227)

J. Cell Biol.166, 61–71.
Mutations in sticky lead to defective organization of the contractile ring during cytokinesis and are enhanced by Rho and suppressed by Rac.2004

D’Avino, P.P., Savoian, M.S., and Glover, D.M.

Notes: The entire sti gene (~7kb) of Drosophila was PCR cloned into the pGEM®-T Vector. The cloned sequences were then sequenced and analyzed for mutations. The researchers also used the T7 RiboMAX™ Large Scale RNA Production System to create dsRNAs from PCR-amplified regions of the sti and GFP coding regions. TransFast™ Transfection Reagent was used to transfect 2 x 106 Schneider S2 cells with 10 μg of dsRNA in a 35mm Petri dish. The cells were fixed after transfection and examined for multinucleate cells, or immunocytochemically stained for actin and anillin. (3259)

Notes: The Wheat Germ Extract was used to demonstrate that the 3’ end of a viral mRNA acts as a tRNA-like structure (TLS). The tRNA-like structure was shown to accept a [H3]valine residue in the Wheat Germ Extract. The turnip yellow mosaic virus (TYMV) TLS structure was also shown to be able to function with ribosomes by donating [H3]valine in translation assays using Wheat Germ Extract with TYMV RNA but not BMV or AMV RNA. RNAs to be used as templates in the reactions were transcribed with the T7 RiboMAX™ Large Scale RNA Production System. After transcription DNA templates were removed with RQ1 RNase-Free DNase. (3386)

Notes: The authors examined the effect of blocking the mRNA of RGR-1, a factor implicated in activation of transcription, in C. elegans embryos. RNA interference (RNAi) was performed by synthesizing sense and antisense rgr-1 cDNA using Promega's RiboMAX™ Large Scale RNA Production System. The effects of RNAi on the cell were examined by looking at the arrested embryos by microscopy and immunostaining. (2559)

Notes: Template DNAs encoding c-Fos (118–211 amino acids) and c-Jun (216–318 amino acids) were generated by PCR, purified and then in vitro transcribed using the RiboMAX™ Large Scale RNA Production System. Following transcription, the RNA was purified and then translated in a wheat germ extract system supplemented with FluoroTect GreenLys tRNA. The fluorescently labeled proteins generated by the translation reaction were run on a 16.5% Tricine-SDS–PAGE and analyzed with a Molecular Imager FX. Labeling efficiency was calculated by measuring total protein via T7-antibody and determining the amount of fluorescently-labeled protein by fluorescence correlation spectroscopy (FCS). (3084)

Notes: Control regions of a naturally occurring dicistronic promoter from Cricket Paralysis Virus (CrPV) were cloned into a previously described dicistronic reporter vector coding for both Fluc and Rluc. The reporter was used either for in vitro translation from RiboMAX™-produced uncapped RNA (RiboMAX™ Large-Scale RNA Production System) using either an RRL or WGE system, and measuring the translation using the Dual-Luciferase® Reporter Assay System, or for transfection into SL-2 cells. RNA was also used for transfection of the SL2 cells, and luciferase activities were measured using DLR. Luciferase readings are reported as ratio of Fluc activity to Rluc activity, with the no insert vector ratio set to 1. (2147)

Notes: The RiboMAX™ Large Scale RNA Production System was used to in vitro transcribe the five plasmids altogether containing the complete genome of Daniel's strain of Theiler's virus. The purified RNA was mixed and electroporated into BHK-21 cells for the in vivo assembly of recombinant virus particles. By using this method, regions within one of the plasmids could be mutated more easily, and recombinant mutant viruses easily obtained. (0769)

Notes: PKA was used to activate channels for single channel patch-clamp recordings to compare single channel conductance of the V470 and M470 alleles of CFTR (cystic fibrosis transmembrane conductance regulator) proteins. PKA was used at a concentration of 75 nM. The RiboMAX™ Large-Scale RNA Production System-T7 was used to transcribe CFTR RNA for injection into Xenopus oocytes. (2167)

Biochemistry37, 4985-4992.
The binding site of a specific aminoglycoside binding RNA molecule1998

Cho, J., Hamasaki, K., Rando, R.R.

Notes: The RiboMAX™ Large Scale RNA Synthesis System was used to produce various 40mer RNA transcripts from synthetic oligonucleotide templates. The transcribed RNAs were purified from 18% acrylamide gels and renatured prior to use. The transcripts were used to determine physical characteristics of the interaction of aminoglycoside and the RNA. (1342)

J. Biol. Chem.272, 20756-20763.
Characterization of point mutations in patients with X-linked ichthyosis. Effects On the structure and function of the steroid sulfatase protein.1997

Alperin, E. S. and Shapiro, L. J.

Notes: Transcripts were produced using the RiboMAX™ Large Scale RNA Production System and expressed in the Rabbit Reticulocyte Lysate System with or without Canine Pancreatic Microsomal Membranes (CMMs). Proteinase K protection assays were performed with proteins expressed in the presence of the membranes with and without Triton® X-100 solubilization of the membranes. (1509)

J. Biol. Chem.272(33), 20756-20763.
Characterization of point mutations in patients with X-linked ichthyosis: Effects on the structure and function of the steroid sulfatase protein.1997

Alperin, E.S. and Shapiro, L.J.

Notes: The ribonuclease inhibitor was used to protect RNA during the reverse transcriptase reaction for RT-PCR. The RNasin® Ribonuclease Inhibitor is also used in the RiboMAX® System for RNA stabilization during in vitro transcription. Transcripts were produce with the RiboMAX® System and expresed in Rabbit Lysate with or without Canine Microsomal Membranes. Proteinase K protection assays are performed with proteins expressed in the presence of the membranes with and without Triton X-100 solubilization of the membranes. The proteinase K digests were stopped by the addition of PMSF and immediately loading the sample into 100°C SDS Sample buffer. (1670)

Notes: The Altered Sites® II in vitro Mutagenesis System was used to produce three single mutants by the single-stranded DNA protocol. The mutants were in vitro transcribed with the RiboMAX® Large Scale RNA Production System in the presence of a cap analog. The capped RNA was microinjected into Xenopus oocytes for expression. (1007)

Notes: The CD69 transcripts were prepared with the T3 RiboMAX® System and translated with the Flexi® System in the presence of the membranes. After 60 minutes at 30°C, the translation reaction was layered onto a sucrose solution and centrifuged at 100,000 x g for 20min. The supernatant was discarded and the pellet was resuspended in SDS-PAGE sample buffer or digested with endoglycosidase H. (1601)

Notes: The RiboMAX™ Large Scale RNA Production System was used to make reference RNA for competitive/quantitative RT-PCR. This is an informative paper about how to perform quantitative RT-PCR and ensure the reaction is linear. The authors developed a series of equations to compensate for nonequality of amplification of different templates. (1461)

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